Detection of target molecules or analytes in biological samples is central to diagnostic monitoring of health and disease. Key requirements of analyte detection are specificity and sensitivity, particularly when the target molecule or analyte is in a limiting amount or concentration in a biological sample.
Typically, specificity is provided by monoclonal antibodies which specifically bind the analyte. Sensitivity is typically provided by a label bound to the specific antibody, or to a secondary antibody which assists detection of relatively low levels of analyte. This type of diagnostic approach has become well known and widely used in the enzyme-linked immunosorbent sandwich assay (ELISA) format. In some cases, enzyme amplification can even further improve sensitivity such as by using a product of a proenzyme cleavage reaction catalyzing the same reaction. Some examples of such “autocatalytic” enzymes are trypsinogen, pepsinogen, or the blood coagulation factor XII. However, in relation to specificity antibodies are relatively expensive and can be difficult to produce with sufficient specificity for some analytes. Polyclonal antibodies also suffer from the same shortcomings and are even more difficult to produce and purify on a large scale.
Current methods to detect specific target molecules and analytes for either prognostic or diagnostic purposes suffer from a number of limitations which significantly restrict their widespread application in clinical, peri-operative and point-of-care settings. Most importantly, the vast majority of diagnostic assays require a significant level of technical expertise and a panel of expensive and specific reagents (most notably monoclonal antibodies) along with elaborate biomedical infrastructures which are rarely available outside specialized laboratory environments. For instance, ELISAs—the gold standard for detecting specific analytes in complex biological samples—rely on the selective capture of a target analyte on a solid surface which in turn is detected with a second affinity reagent that is specific for the target analyte. ELISAs also feature extensive incubation and washing steps which are generally time consuming and difficult to standardize as the number of successive steps frequently introduces significant variation across different procedures, operators and laboratories making quantitative comparisons difficult. In addition, it is often desirable to measure the active state of a target analyte, and not just detect its presence as is frequently only possible with ELISAs. This particularly applies to proteases whose activity is tightly regulated as they carry out critical functions in a range of important physiological processes, and are frequently de-regulated in different disease states including cancer (e.g. matrix metalloproteinases), infectious disease (e.g. pathogen specific proteases) and cardiac disorders (e.g. components of the blood coagulation cascade).
For instance, blood coagulation assays feature some of the most widely established protease assay platforms which are routinely applied in clinical laboratories to diagnose aberrant coagulation processes caused by drugs or underlying pathological processes. The majority of conventional blood screening tests that are used to characterise haemostatic phenotypes (including indirect assay methods used to quantify individual components of the blood coagulation cascade) generally measure the time to form a fibrin clot and almost exclusively relay their effect through multiple components of the blood coagulation cascade, in particular thrombin and the common pathway: e.g. prothrombin time, activated partial thromboplastin time, thrombin and reptilase time and various fibrinogen assays. These assays usually take advantage of the natural amplification power of the blood clotting system; this is however associated with a number of disadvantages: For example, the dependence on multiple clotting factors for a particular read-out limits their precision as variations in the genetic background of an individual generally make it hard to deconvolute the contribution of one particular component of the blood coagulation cascade to a given haemostatic phenotype observed in the clinic. Similarly, regulatory mechanisms in protease signalling networks and physiological variations in the components of the coagulation cascade (e.g. in the presence or absence of drugs etc.) make it difficult to translate assay data into targeted treatment regimes in the clinic as there is no clear-cut correlation between cause and effect at the molecular level.
Beyond blood coagulation, proteases are also increasingly recognized to carry out critical functions in a number of disease processes; these are expected to open new avenues for diagnostic and therapeutic regimes. Since most proteases are of relatively low abundance and do not readily form part of extended protease signalling networks with intrinsic amplification power (compared with blood coagulation system), the establishment of highly sensitive, specific and easily operable protease assays has remained a challenge to date. Two widely applied protease assays are based on zymography and FRET-based fluorescent assays based on short peptide substrates: Compared to ELISAs, zymography carries the advantage that only active proteases are assayed while inactive zymogens are not. However, zymographic techniques are generally laborious taking up to two days to develop (Kupai, et al., 2010, J Pharmacol Toxicol Methods, 61, 205-9.) and are thus not readily integratable into point-of-care devices. In addition, problems persist with standardisation, sensitivity and data interpretation. More recently, FRET-based fluorescent assays based on highly specific peptide substrates have been developed. Substrate peptides can either be derived from natural substrates or can be artificially engineered for greater specificity. Nevertheless, limited sensitivities remain especially if the target protease is present in limiting amounts: e.g. the best substrates for matrix metalloproteinases developed to date take about two hours to develop in cell culture and tolerate only limited amounts of plasma (Jabaiah & Daugherty, 2011, Chem Biol 18, 392-401) while screens do not readily select against promiscuous substrate specificities. From a biophysical point of view, the lack of specificity and selectivity of peptide based substrates can largely be attributed to the limited surface area which any given peptide can provide towards molecular recognition as well as the lack of biomolecular signal amplification which is a trademark of biological signalling systems.